Sustained long-term antibody levels are the cornerstone of protective immunity, yet the mechanism(s) by which such titers are durably maintained remains unclear. The prevailing paradigm is this occurs via continuous antigen-independent antibody production by a subset of long-lived plasma cells (LLPC) that survive within a limited number of specific niches in the bone marrow (BM). The paradigm's central tenets however, that BM LLPC are a separate subset defined by intrinsically distinct biology and that they in fact contribute to long-term antibody titers, have not been directly demonstrated. We have recently reported (Rozanski et al, JEM July, 2011) that long-term humoral immunity is dependent on plasma cell-intrinsic function of CD28 (best characterized as the prototypic T cell costimulatory receptor), which selectively supports the survival of BM LLPC but not splenic short-lived PC (SLPC). Although LLPC and SLPC both expressed CD28, CD28 signaling and enhanced survival only occurred in the LLPC (and unlike T cells did not require an exogenous signal 1). In vivo, even with sufficient T cell help the loss of CD28 in the plasma cells (or the global loss of the CD28 ligands CD80 or CD86) caused significant loss of the LLPC population, reduction of the half-life of LLPC survival in the BM from 426 to 63 days, and inability to maintain antibody titers long-term - without having any effect on SLPC populations. On the microenvironment/niche side, we found that CD80+ dendritic cells (DC) were in direct contact with LLPC within the BM, that myeloid DC supported long-term LLPC survival in a CD28-dependent fashion in vitro, and that backsignaling via CD80 and CD86 ligation induced the DC to produce the pro-PC survival cytokine IL-6 that was unexpectedly essential for sustaining immunoglobulin (Ig) production in vitro. Altogether, these findings establish the existence of the distinct BM LLPC subset that is necessary to sustain antibody titers, and uncover a previously unrecognized but essential role for PC-intrinsic CD28 function in the longevity of plasma cells and humoral immunity. While previous studies have clearly defined a role for CD28 in both protective and pathogenic (e.g. autoimmune) antibody responses, this has been almost entirely attributed to costimulation of helper T cell activation. Reconsideration of this literature raises he important question of how much of the humoral response is due to CD28 function in T cells vs. plasma cells. Thus we believe that PC-intrinsic CD28 function plays a central role in plasma cell biology and the generation of durable humoral immunity, but has been almost entirely uncharacterized in this context. The overall goal of this proposal is to define the specific mechanisms by which CD28 activation regulates PC survival, function, and the development of humoral immune responses. The Aims of this proposal are to: 1). Define the signal transduction pathways and molecular targets downstream of CD28 activation that modulate PC survival and function, 2). Characterize the CD80/CD86+ LLPC BM stromal niche, and 3). Characterize the role of PC vs. T cell intrinsic CD28 function in humoral immune responses in mice conditionally knocked out for CD28 in the T or B cell lineage.